Vaccine for equine rhinopneumonitis

ABSTRACT

Live Bovine Herpesvirus A.T.C.C. VR-2003 is employed in vaccination of horses to confer immunity to infection by Equid Herpesvirus Type 1.

BACKGROUND

The present invention relates generally to novel immunological materialsand methods, and more particularly to treatment of horses to conferimmunity to infection by Equid Herpesvirus Type 1.

Equine rhinopneumonitis is an infectious disease of horses caused by aviral strain known as Equid Herpesvirus Type 1 (hereafter, "EHV 1"). Thedisease state is variously characterized by upper respiratory infection,by abortion in pregnant mares, and by neuro-musclar symptoms such asataxia and paresis. While EHV 1 is known to produce abortion inhamsters, calves are reported to be refractory to infection with thisvirus.

Commercial vaccines comprising hamster-adapted and tissue cultured liveEHV 1 virus have been generally inadequate in controlling the spread ofthe disease. More particularly, administration of prior art vaccines hasfrequently been accompanied by incidences of abortion in maresvaccinated during pregnancy and by infection of contact animals byvaccine virus shed nasally after vaccination. As a result, vaccinationof pregnant mares is avoided as is vaccination of horses expected tocome in contact with unvaccinated mares. There therefore exists a needfor vaccine materials and methods which confer immunity to infection byEHV 1 but which are safe when administered to pregnant mares and whichdo not give rise to shedding of infective vaccine virus aftervaccination.

This invention has its background in part in the inventor's discoveryand isolation of a herpesvirus from the lung of an aborted Herefordfetus. The isolation of this herpesvirus is one of two known isolationsfrom the bovine species. The specific virus isolated by the inventor wasshown by him to possess physical properties common to those of theherpesviruses, e.g., typical herpesvirus morphology as confirmed byelectron microscopic studies. The virus was found to be sensitive tochloroform and its replication was inhibited by 5-iodo-deoxyuridine.Initial serum neutralization tests using diluted typing sera specificfor EHV 1 showed the newly-isolated viral strain (designated "BovineHerpesvirus No. 1247") to be distinct from, inter alia, the equineherpesvirus responsible for equine rhinopneumonitis. See, generally,Crandell, et al, "Theriogenology", 6, pp. 1-19, (1976). Subsequentindependent studies of samples of the new virus showed, however, theexistence of serologic relation with other viruses, and specificallywith EHV 1. See, Kanitz, et al, "Proceedings, 19th Meeting AmericanAssociation of Veterinary Laboratory Diagnosticians," pp. 1-12 (Nov.1976) and Smith, "Proceedings, 80th, Meeting, U.S. Animal HealthAssociation," pp. 149-158, (1976).

In a comparative study of Bovine Herpesvirus No. 1247 and EHV 1, theinventor ascertained that the bovine virus was in fact nasally infectivein horses, generating a mild rhinitis and increase in rectaltemperature. The inoculated ponies developed measurable serumneutralizing antibodies to the vaccine virus as well as to the equinevirus (EHV 1).

BRIEF SUMMARY

According to the present invention vaccines are prepared which comprisedBovine Herpesvirus A.T.C.C. No. VR-2003 in dosage amounts of from about10³ to about 10⁶ TCID₅₀ dispersed in an immunologically acceptablecarrier. Mutant forms of A.T.C.C. No. VR-2003 prepared, e.g., bypropagation at reduced temperatures and/or in the presence of mutagenicchemicals, are also expected to be useful. Vaccines so prepared areadministered to horses intranasally or parenterally (intramuscularly orsubcutaneously) and confer immunity to infection against EHV 1.

Horses vaccinated according to the methods of the present inventiondevelop enhanced antibody titers to EHV 1 and post-vaccination challengewith EHV 1 gives rise to substantially diminished symptoms of infection.Vaccination of pregnant mares does not give rise to abortion. Finally,vaccine virus is not shed by vaccinated animals, therefore it does notappear to be a problem for contact animals.

DETAILED DESCRIPTION

The Bovine Herpesvirus strain employed in the practice of the presentinvention possesses biochemical and biophysical properties common tomembers of the herpesvirus group. Its replication is inhibited by5-iodo-deoxyuridine, indicating that its essential nucleic acid is DNA.The virus is sensitive to chloroform treatment and is heat labile. Virusparticle morphology is similar to the other herpesviruses. [See, e.g.,Wilner, "A Classification of the Major Groups of Human and Other AnimalViruses," Ch. 10, p. 103 (Burgess Pub. Co., Minneapolis, Minn. 4th Ed.1969].

The virus has been deposited by the inventor and is available fromAmerican Type Culture Collection, 12301 Parklawn, Rockville, Md. 20852under the designation A.T.C.C. VR-2003. The deposited viral strain isthe result of sixteen serial passages of the original bovine isolate inprimary bovine embryonic kidney (BEK) cells [Flow Laboratories]maintained on medium MEM [Grand Island Biological Company] plus 2%bovine fetal serum. The culture medium also contained 200 units ofpenicillin, 200 mg of streptomycin, and 2.5 mg of amphotericin B per ml.

The following example illustrates the effectiveness of BovineHerpesvirus A.T.C.C. VR-2003 a live viral component of a vaccine toconfer immunity to infection by EHV 1.

EXAMPLE 1

Animals. Six shetland mare ponies in differing stages of gestation wereemployed.

Cell Cultures. Propagation of viral strains was carried out in equineembryonic kidney (EEK) cells in third and sixth subcultures grown intubes and flasks with Eagle's minimum essential medium (MEM). Pig kidney(PK-15) cells for use in serum neutralization tests were grown in MEMwith nonessential amino acids, glutamine, 0.5% lactalbumin hydrolysate,and 10% fetal bovine serum. All culture media additionally included 200units of penicillin, 200 μg of streptomycin, and 2.5 μg of AmphotericinB per ml.

Sample collection. Nasal swabs for virus isolation were obtained bystreaking the nasal septum with a cotton tipped applicator. Swabs werestored in 1 ml MEM medium at -70° C. until testing. Serum samples forserological tests were collected at time intervals indicated infra.

Virus Isolations. BEK and EEK cell cultures were inoculated with 0.2 mlof MEM fluid containing nasal specimens and observed daily forcytopathic changes characterized by syncytial formations withdestruction of the cell monolayer.

Serum-Virus Neutralization Tests. Reciprocal serum-virus neutralizationtests were performed with EHV 1 by microtiter methods unless otherwiseindicated. Equal amounts of serum and virus were mixed with 200 to 300TCID₅₀ doses of virus and were incubated at 37° C. for one hour beforeadding PK-15 cells. Serum titers are expressed as the reciprocal of thefinal serum dilution in which cytopathic changes were absent after 5days.

Vaccines. Bovine Herpesvirus A.T.C.C. VR-2003 was propagated in quantityin the second subculture of EEK cells described above. The virusinoculated cultures were incubated for 4 days at 36° C. Following onefreeze and thaw cycle, the virus suspension was clarified by low speedcentrifugation. The vaccine (virus) was dispensed into vaccine bottlesand stored at -70° C. until used. The EHV 1 employed was originallyisolated from the liver of an aborted foal and was propagated inquantity after five serial passages in EEK cells.

Procedure. Ponies No. 1 through 5 were inoculated subcutaneously withone 10 ml. dose containing 10⁵.6 TCID₅₀ of Bovine Herpesvirus A.T.C.C.VR-2003 in MEM fluid. Pony No. 6 served as a contact control and wasinoculated with 10 ml of normal culture medium (MEM) in two sites. TheEHV 1challenge virus was administered to ponies No. 2 through 6, 14 daysafter foaling of the last-to-foal mare. The challenge virus wasinoculated intranasally in a 5 ml. dose containing 10⁵.7 TCID₅₀ virus.Sera was collected and subjected to serum neutralization testing beforevaccination, at 14, 21, and 192 or 208 days after vaccination, and 21days after the EHV 1 challenge inoculation. Virus isolations from nasalpassages were obtained on the day of EHV 1 challenge and on each of theeight days following challenge.

Results. Table 1 below provides data concerning the experimental animalsin terms of age, day of gestation at the time of vaccination, and daysafter vaccination when mares foaled. Significantly, there was noincidence of abortion in any of the vaccinated ponies, nor was thereabortion in the control pony.

                  TABLE 1                                                         ______________________________________                                                                        Days after                                    Pony   Age       Day of Gestation                                                                             Vaccination                                   No.    (yrs.)    at Vaccination Mare Foaled                                   ______________________________________                                        1      12        303            40                                            2      7         227            116                                           3      14        319            24                                            4      3         309            34                                            5      4         319            24                                             6*    14        149            194                                           ______________________________________                                         *Unvaccinated Control                                                    

Table 2 below provides data concerning serum neutralization titers toEHV 1 by microtiter method, reported as the reciprocal of endpointdilution. Bracketed values are those obtained by standard tube methods,without complement added, using 0.2 ml inoculum. All but the controlanimal showed significant increases in EHV 1 antibody titers aftervaccination with Bovine Herpesvirus A.T.C.C. VR-2003.

                  TABLE 2                                                         ______________________________________                                        Serum Neutralization Titer                                                                                       Post-                                                        Post-Vaccination Challenge                                  Pony    Pre-      Days             Days                                       No.     Vaccination                                                                             14     21       208  21                                     ______________________________________                                        1       16    [22]    64   64   [710] .sup.(1)                                                                           --                                 2       32    [26]    NT.sup.(2)                                                                         128  [356] 64.sup.(3)                                                                         128                                3       64    [14]    NT   128  [296] 64.sup.(3)                                                                         128                                4        4    [10]    64   128  [1002]                                                                              128  256                                5        4    [12]    64   64   [710] 64   512                                6(control)                                                                             4    [22]     8   4    [22]  16   256                                ______________________________________                                         .sup.(1) Pony No. 1 was sacrificed because of injury.                         .sup.(2) Not Tested.                                                          .sup.(3) Titers are 192 days postvaccination.                            

Table 3 below provides data concerning isolation of virus shed by theponies after EHV 1 challenge. Vaccination with Bovine HerpesvirusA.T.C.C. VR-2003 diminished virus shedding to 1 day indicating that onlyresidual virus (the inoculum) was recovered.

                  TABLE 3                                                         ______________________________________                                        Virus Isolation                                                               Days Post-Challenge                                                           Pony No.                                                                              0      1      2    3    4    5   6    7   8                           ______________________________________                                        2       -      +      -    -    -    -   -    -   -                           3       -      +      -    -    -    -   -    -   -                           4       -      +      -    -    -    -   -    -   -                           5       -      +      -    -    -    -   -    -   -                           6       -      +      +    +    -    -   -    -   -                           ______________________________________                                    

Suitable vaccines prepared according to the present invention containdosage amounts of from about 10³ to about 10⁶ TCID₅₀ of the live virusand preferably about 10⁴ to about 10⁵. Suitable immunologicallyacceptable carriers for the virus include cell culture fluid and astabilizer. Administration of the vaccine according to the methods ofthe invention is preferably by the intranasal route and parenteralroutes including subcutaneous and intramuscular.

As indicated in the foregoing example, it is preferred that BovineHerpesvirus A.T.C.C. VR-2003 be propagated in cell cultures of equineorigin prior to vaccine preparation. This is due to the observedenhanced replication of the virus in cells of equine origin as opposedto bovine cell cultures in which it is maintained.

It is within the contemplation of the present invention that BovineHerpesvirus A.T.C.C. VR-2003 may be subjected to multiple serialpassages in cell cultures of equine, bovine, or other species prior touse in vaccine preparations. It is also contemplated that mutant formsof A.T.C.C. VR-2003 will be useful in practice of the invention.Modifying procedures including propogation of the virus at lowertemperatures and/or with mutagenic chemical treatments are expected topermit selection of more temperature-sensitive viruses suitable asvaccine strains. Such procedures are expected to decrease virulence orinfectivity without substantially diminishing the antigenicity of theA.T.C.C. VR-2003 strain.

Numerous modifications and variations of the above disclosed methods andmaterials of the invention are expected to occur to those skilled in theart. Consequently only such limitations as appear in the appended claimsshould be placed on the invention.

What is claimed is:
 1. A method for treatment of a horse to conferimmunity to infection by Equid Herpesvirus Type 1, said methodconsisting of administrating a vaccine comprising an immunologicallyeffective amount of the live Bovine Herpesvirus A.T.C.C. No. VR-2003. 2.The method of claim 1 wherein the dose of live virus administered isfrom about 10³ to about 10⁶ TCID₅₀.
 3. The method of claim 1 wherein themode of administration of said vaccine is intranasal.
 4. The method ofclaim 1 wherein the mode of administration is parenteral.
 5. A vaccinesuitable for use in conferring upon horses immunity to infection byEquid Herpesvirus Type 1, said vaccine comprising, in unit dosage form,from about 10³ to about 10⁶ TCID₅₀ of Bovine Herpesvirus A.T.C.C.VR-2003 in combination with an immunologically acceptable carrier.